Several microorganisms are able to use an aromatic hydrocarbon as a sole source of carbon and energy. This capability results from highly specialized pathways that convert the aromatic hydrocarbon to intermediates in the Krebs cycle. Initially, the aromatic hydrocarbon is converted to catechol or a substituted catechol. Subsequently, the catecholic compound is processed to the Krebs cycle by one of the many so- called meta-fission pathways. The battery of enzymes comprising these meta-fission pathways are rich in mechanistic, structural, and evolutionary questions and present an attractive strategy for the degradation of aromatic hydrocarbons in the environment. The long term goal of this research is to apply techniques from protein chemistry, molecular biology, and x-ray crystallography to study thoroughly the mechanism, structure, and evolution of the enzymes in two meta-fission pathways - the catechol and homoprotocatechuate meta-fission pathways. During this funding period, we will continue our studies on 4- oxalocrotonate tautomerase (4-OT), 4-oxalocrotonate decarboxylase (4-OD), and vinylpyruvate hydratase (VPH). All are in the catechol meta-fission pathway. Our major specific aims, listed in the order of priority, during this funding period will be to: (l) Examine further the "anchoring" hypothesis for 4-OT using substrate analogs; (2) Characterize the behavior of 4-OT with a potential acetylenic mechanism-based inhibitor and a potential affinity label; (3) Examine the role of proline-1 in the mechanism of 4-OT by the chemical synthesis and characterization of mutants; (4) Continue NMR studies of 4-OT as a prelude to a solution structure; (5) Determine the 13C kinetic isotope effects on the non-enzymatic and the 4-OD-mediated decarboxylation of 2-oxo-3-hexenedioate and compare the results to those previously determined for the decarboxylation of oxalacetate; (6) Determine the steric course of proposed allylic isomerization and a hydration reaction catalyzed by VPH if the configuration of the intermediate can be established; (7) Assign the stereochemistry at C-5 of the product of the VPH reaction; and (8) Examine the behavior of VPH with the acetylenic analog of its substrate. The proposed studies will address key mechanistic and evolutionary questions raised by what appears to be a common strategy used by the various meta-fission pathways to degrade catecholic compounds. These studies also have implications for the understanding of basic enzymological processes, enol and dienol chemistry, and the evolution of enzyme specificity and catabolic pathways. The genetic analysis coupled with the enzymological studies proposed herein set the stage to understand the mechanism as crystal structures become available and to probe the role of specific active site residues in catalysis and specificity through future site-directed mutagenesis. In addition to addressing these intellectual questions, these studies may ultimately have applications toward a solution of the incumbent toxic waste problem.